
==== Front
J Exp Clin Assist ReprodJournal of Experimental & Clinical Assisted Reproduction1743-1050BioMed Central London 1743-1050-2-31570307410.1186/1743-1050-2-3Short ReportCorrelation of sperm penetration assay score with polyspermy rate in in-vitro fertilization Aoki Vincent W 123vincent.aoki@hsc.utah.eduPeterson C Matthew 4c.matthew.peterson@hsc.utah.eduParker-Jones Kirtly 4kirtly.jones@hsc.utah.eduHatasaka Harry H 4harry.hatasaka@hsc.utah.eduGibson Mark 4mark.gibson@hsc.utah.eduHuang Ivan 4ivan.huang@hsc.utah.eduCarrell Douglas T 1234douglas.carrell@hsc.utah.edu1 Andrology and IVF Laboratories, University of Utah School of Medicine, Salt Lake City, UT, 84108, USA2 Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT, 84132, USA3 Department of Physiology, University of Utah School of Medicine, Salt Lake City, UT, 84132, USA4 Department of Obstetrics and Gynecology, University of Utah School of Medicine, Salt Lake City, UT, 84132, USA2005 9 2 2005 2 3 3 15 12 2004 9 2 2005 Copyright © 2005 Aoki et al; licensee BioMed Central Ltd.2005Aoki et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background
The sperm penetration assay (SPA) is used to predict the fertilizing capacity of sperm. Thus, some programs rely on SPA scores to formulate insemination plans in conjunction with in-vitro fertilization (IVF) cycles. The purpose of this study was to evaluate if a relationship exists between SPA scores and polyspermy rates during conventional IVF cycles.

Methods
A total of 1350 consecutive IVF patients using conventional IVF insemination were evaluated in the study. Oocytes were inseminated three hours post-retrieval by the addition of 150,000 to 300,000 progressively motile sperm. Approximately 18 hours after insemination, the oocytes were evaluated for fertilization by the visualization of pronuclei. The presence of three or more pronuclei was indicative of polyspermy. Polyspermy rates, fertilization success, embryo quality, and pregnancy rates were analyzed retrospectively to evaluate their relationship with SPA score, count, motility, number of progressively motile sperm inseminated, oocyte pre-insemination incubation time, patient age, and diagnosis.

Results
A significant positive relationship was observed between SPA score and polyspermy rate (rs = 0.10, p < 0.05). Patients with a normal SPA score had significantly higher polyspermy rates than those with abnormal SPA scores (6.3% ± 1.5% vs. 2.0% ± 0.7%, p < 0.05). Fertilization percentage was significantly lower in the group with severely abnormal SPA scores versus all other SPA groups (57.5% ± 2.1% vs. 70.2% ± 1.3%, p < 0.005). Although embryo quality was not affected, both clinical pregnancy and implantation rates improved slightly as SPA score increased. In addition, there was a decrease in the rate of spontaneous abortion as SPA score increased.

Conclusions
These data indicate SPA score is positively correlated with polyspermy rates and IVF fertilization percentage. Additionally, there is a slight increase in clinical pregnancy rates, and embryo implantation rates with increased SPA. Furthermore, there is a slight decrease in spontaneous abortions rates related to increased SPA.
==== Body
Findings
The sperm penetration assay (SPA) is used to evaluate the fertilizing capacity of human spermatozoa [1]. Some in-vitro fertilization (IVF) programs rely on the SPA to formulate their insemination plans in conjunction with IVF cycles [1,2]. Couples with normal SPA scores normally undergo conventional IVF insemination in contrast with intracytoplasmic sperm injection (ICSI), which is used for those with a diminished SPA.

Over the past decades success rates have steadily improved for human IVF embryo transfer programs. Recent advances in ovulation induction, micromanipulation, culturing conditions, and media formulations have fostered a technological revolution for IVF leading into the 21'st century (Pool, 2002). However, many common problems still remain.

One common problem associated with conventional IVF insemination is polyspermic fertilization when more than one sperm successfully penetrates and fertilizes the oocyte. These pre-embryos are considered abnormal and typically discarded. Thus, increased rates of polyspermy ultimately result in a reduced number of embryos.

Studies have been conducted that evaluate the factors associated with human IVF polyspermy [3-5]. However, most of the reports are based on data over a decade old. Recent improvements in IVF techniques may alter these relationships. Furthermore, no study has evaluated the relationship between sperm SPA score and incidence of polyspermy in human IVF.

The purpose of this study was to evaluate if a relationship exists between SPA scores and polyspermy rates during IVF cycles utilizing conventional insemination. We also evaluated the relationship between SPA score and fertilization rate, embryo quality, and pregnancy rate.

After Institutional Review Board approval, we conducted a retrospective study of 1350 consecutive IVF patients using conventional IVF insemination. Intracytoplasmic sperm injection (ICSI) patients were excluded from the study. The fertilizing capacity of each patient was assessed with the SPA. The SPA was performed on patients undergoing IVF using techniques previously described [6]. The SPA was performed on patients less than sixty days prior to the IVF cycle. The SPA score reflects the percentage of eggs successfully penetrated by the patient's sperm. SPA patients were stratified into 4 groups according to SPA score (Group 1: severely abnormal SPA (< 10% penetration), n = 182; Group 2: abnormal SPA (10–19% penetration), n = 368; Group 3: normal SPA (20 – 29% penetration), n = 404; Group 4: high-normal SPA (> 30% penetration), n = 396).

During the IVF cycle ovarian stimulation was performed using standard techniques of gonadotrophin-releasing hormone (GnRH) agonist down-regulation combined with controlled ovarian stimulation using a combination of recombinant follicle stimulating hormone (rFSH) and urinary-derived gonadotropin stimulation. Ovarian follicles were aspirated using a trans-vaginal ultrasound-guided needle.

Oocytes were inseminated three-hours post-retrieval by the addition of 150,000 to 300,000 progressively motile sperm. Approximately 18-hours post-insemination, the oocytes were evaluated for fertilization by visualization of pronuclei. The presence of three or more pronuclei was indicative of polyspermy in which case the fertilized oocyte was discarded. Embryos were cultured in HTF medium and transferred 72-hours post-retrieval. Embryo quality was assessed using a previously reported embryo scoring system that took into account the number of cells present and the level of cellular fragmentation [7].

Polyspermy rates, fertilization success, embryo quality, and pregnancy rates were analyzed retrospectively with respect to their relationship with SPA score. The correlation between SPA score and polyspermy was conducted using Spearman's correlation coefficient. Additionally, polyspermy rates, fertilization percentage, and embryo quality for the different SPA groups were analyzed for statistical difference using Kruskal-Wallis analysis. Lastly, pregnancy rates in the different groups were evaluated with a Chi-square analysis.

A significant positive relationship was observed between SPA score and polyspermy rates in 1350 IVF patients undergoing conventional IVF insemination (rs = 0.10, p < 0.05). Patients with normal SPA scores (Group 3) had significantly elevated polyspermy rates over those with severely abnormal SPA scores (6.3 ± 1.5 – 113/1,791 vs. 2.0 ± 0.7 – 20/1,020, p < 0.05, Table 1, please see file "Table1-Aoki et al.doc", Additional file 1). Conventional IVF fertilization rate was significantly lower in patients with severely abnormal SPA (57.5 ± 2.1 – 1,135/1,974) vs. all other patients (70.2 ± 1.3 – 8,519/12,135; p < 0.005, Table 1, please see file "Table1-Aoki et al.doc", Additional file 1).

Embryo quality showed no relationship to the SPA (Table 1, please see file "Table1-Aoki et al.doc" Additional file 1). Clinical pregnancy and implantation rates were increased in Group 4 (50.8% – 202/396 and 27.4% – 343/1255, respectively) versus Group 1 (31.6% – 58/182 and 15.7% – 96/612, respectively, p < 0.05) but no significant relationship between SPA and IVF outcome was observed throughout the entire study population (Table 1, please see file "Table1-Aoki et al.doc" Additional file 1). Similarly, there was a decrease in the rate of spontaneous abortion in Group 4 (14.7% – 30/202) versus Group 1 (31.3% – 18/58), although no significant relationship was observed within the entire population (Table 1, please see file "Table1-Aoki et al.doc" Additional file 1).

No relationships were observed between IVF polyspermy rates and maternal age, maternal diagnosis, amount of progressively motile sperm added, sperm morphology, ovarian stimulation protocol, or post-oocyte retrieval pre-insemination incubation time. Additionally, confounder analysis indicated the SPA groups were similar with respect to paternal age, maternal age and IVF diagnosis. Therefore, no standardization of the data with respect to these variables was required.

This is the first report to correlate SPA score with IVF polyspermy rates. Significantly elevated IVF polyspermy rates were observed in patients with normal SPA scores. Previous reports have established a relationship between IVF polyspermy and oocyte age/maturation, state of the zona pellucida, number of progressively motile sperm used for insemination, oviductal cytokine expression, and in-vitro culture conditions such as pH, temperature, and media supplementation [5].

Unlike other reports, we did not find a relationship between the number of progressively motile sperm used for insemination and IVF polyspermy rates. This discrepancy is most likely due to the narrow range of sperm number we used for insemination (150,000 to 300,000) compared with the studies of van der Ven et al. who used 500,000 to 1.5 *106 sperm [4]. Our results are consistent with Ho et al. who found no relationship between these variables [3]. However, the data still suggest minimizing the number of sperm added for IVF insemination in patients with increased SPA scores.

Poly-pronuclear formation in in-vitro fertilized eggs usually arises from polyspermic fertilization but may also be a product of second polar body retention [8]. Thus, one potential pitfall of associating poly-pronuclear fertilized oocytes with polyspermy is the uncertainty related to second polar body retention. However, a recent report showed only a small percentage of fertilized oocytes with three-pronuclei (2.5%) arise from second polar body retention. The findings of this study validate our study design of categorizing poly-pronuclear fertilized oocytes as polyspermic. Moreover, we would expect the contribution of second polar body retention to be consistent throughout the SPA groups negating any confounding effects.

Consistent with other reports, the SPA was correlated with fertilization percentage [1,6]. Furthermore, clinical pregnancy and implantation rates did show improvement in patients with a high-normal SPA versus those with a severely abnormal SPA. In addition, spontaneous abortion rates were significantly different between these two groups. This data strongly suggests ICSI may be an attractive alternative for patients with severely affected penetration ability.

Done correctly, the SPA provides a reliable assessment of the fertilizing ability of human spermatozoa with very low false negative rates (< 0.03%) and serves a valuable tool for clinicians to treat infertility patients with the appropriate modality [9]. The SPA is particularly useful in light of recent concerns about ICSI and imprinting diseases [10-12]. These concerns have led to the recommendation that IVF clinicians should be careful to employ the technique only when necessary and the SPA provides a prognostic tool to appropriately make that decision. Based on these findings, it may be valuable for individual IVF programs to re-evaluate the clinical utility of SPA testing. The SPA may prove valuable for clinicians to avoid increased amounts of polyspermy and unnecessary wasting of oocytes. Meanwhile, the SPA does not appear to be a reliable indicator of IVF embryo quality or pregnancy rates.

Authors' Contributions
VWA designed the study, carried out statistical evaluation, and was the primary author of the manuscript. CMP, KPJ, HHH, MG, and IH were involved in management of the IVF cases and collection of the clinical data. DTC was responsible for the implementation of the study, administrative requirements including IRB approval, statistical evaluation of the data, and preparation of the manuscript.

Supplementary Material
Additional File 1
Table 1. Relationship between SPA and IVF outcome measures

Click here for file
==== Refs
Carrell DT  Semen analysis at the turn of the century: an evaluation of potential uses of new sperm function assays Arch Androl 2000 44 65 75 10690767 10.1080/014850100262434 
Zahalsky MP Zoltan E Medley N Nagler HM  Morphology and the sperm penetration assay Fertil Steril 2003 79 39 41 12524061 10.1016/S0015-0282(02)04412-6 
Ho PC Yeung WS Chan YF So WW Chan ST  Factors affecting the incidence of polyploidy in a human in vitro fertilization program Int J Fertil Menopausal Stud 1994 39 14 19 8167675 
van der Ven HH Al-Hasani S Diedrich K Hamerich U Lehmann F Krebs D  Polyspermy in in vitro fertilization of human oocytes: frequency and possible causes Ann N Y Acad Sci 1985 442 88 95 3860066 
Wang WH Day BN Wu GM  How does polyspermy happen in mammalian oocytes? Microsc Res Tech 2003 61 335 341 12811738 10.1002/jemt.10346 
Carrell DT Bradshaw WS Jones KP Middleton RG Peterson CM Urry RL  An evaluation of various treatments to increase sperm penetration capacity for potential use in an in vitro fertilization program Fertil Steril 1992 57 134 138 1730307 
Carrell DT Peterson CM Jones KP Hatasaka HH Udoff LC Cornwell CE Thorp C Kuneck P Erickson L Campbell B  A simplified coculture system using homologous, attached cumulus tissue results in improved human embryo morphology and pregnancy rates during in vitro fertilization Journal of Assisted Reproduction and Genetics 1999 16 344 349 10459515 10.1023/A:1020533711711 
Porter R Han T Tucker MJ Graham J Liebermann J Sills ES  Estimation of second polar body retention rate after conventional insemination and intracytoplasmic sperm injection: in vitro observations from more than 5000 human oocytes J Assist Reprod Genet 2003 20 371 376 14531648 10.1023/A:1025481011680 
Carrell DT Udoff LT Hatasaka HH Jones KP Peterson CM  Predictability / variability of a modified sperm penetration assay (SPA): review of 1033 patients undergoing zona-free hamster oocyte sperm penetration analyses / IVF Adv Reprod 1998 1 45 56 
Maher ER Brueton LA Bowdin SC Luharia A Cooper W Cole TR Macdonald F Sampson JR Barratt CL Reik W Hawkins MM  Beckwith-Wiedemann syndrome and assisted reproduction technology (ART) J Med Genet 2003 40 62 64 12525545 10.1136/jmg.40.1.62 
Gosden R Trasler J Lucifero D Faddy M  Rare congenital disorders, imprinted genes, and assisted reproductive technology Lancet 2003 361 1975 1977 12801753 10.1016/S0140-6736(03)13592-1 
DeBaun MR Niemitz EL Feinberg AP  Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19 Am J Hum Genet 2003 72 156 160 12439823 10.1086/346031

